Relaxin-3 (also known as INSL7) has been found to be a ligand for the G-protein coupled receptors (GPCRs) GPCR135 and GPCR142. See U.S. Provisional Application No. 60/493,941, filed 7 Aug. 2003, and International Application No. PCT/US2004/005666, filed 25 Feb. 2004, the disclosures of which are incorporated by reference herein.
GPCRs are transmembrane receptor proteins that are responsible for the transduction of a diverse array of extracellular signals, including hormones, neurotransmitters, peptides, lipids, ions, light, odorants, nucleotides, fatty acid derivatives, and other chemical mediators. See, e.g., WIPO Publication No. WO 02/00719. GPCRs are of particular importance to drug discovery because they have been established as excellent drug targets: they are the targets of 50% of marketed drugs. An increasing number of diseases have been found to be associated with GPCRs. Drugs targeting GPCRs have been used to treat a wide range of disorders from cardiovascular to gastrointestinal to CNS and others (Wilson et al., 1998, British J. of Pharmacology, 125:1387-1392).
The GPCR-mediated signal transduction event is often initiated upon binding of a specific ligand to the GPCR. Each GPCR is composed of an extracellular N-terminal domain, seven distinct transmembrane segments, and an intracellular C-terminal domain. Binding of the ligand to an extracellular N-terminal domain, transmembrane domain, or intracellular loop of a GPCR results in a conformational change that leads to activation of intracellular heterotrimeric GTP-binding proteins (G proteins) associated with the GPCR. These activated G proteins in turn mediate a variety of intracellular responses that regulate cell physiology. Therefore, the ligand provides means of elucidating the physiological function of the GPCR as well as methods of screening for compounds that regulate the signal transduction activity of the GPCR.
At present, only about 200 GPCRs are classified as known GPCRs that are activated by around 70 known ligands. Through sequence analyses, it was discovered that GPCRs belong to one of the largest superfamilies of the human genome: evaluated at over 1000 genes encoding GPCRs (Civelli et al., 2001, Trends in Neurosciences, 24:230-237). A large number of putative GPCRs are described as orphan receptors because their natural ligands are unknown. Some of these uncharacterized orphan GPCRs may be useful as therapeutic targets. The identification of the specific ligand to a GPCR is the key to harnessing the potential therapeutic benefits of these orphan GPCRs (Howard et al., 2001, Trends in Pharmacological Sciences, 22:132-140).
As noted above, relaxin-3 (also known as INSL7) has been found to be a ligand for GPCR135 as well as for GPCR142. See Liu et al., 2003, Journal of Biological Chemistry, 278:50754-50764; and Liu et al., 2003, Journal of Biological Chemistry, 278:50765-50770. Relaxin-3 is a member of the insulin/relaxin superfamily. Members in this family are characterized by peptide subunits (A-chain and B-chain) linked by three disulfide bonds. Two of the three disulfide bonds are inter-subunit bonds and another one is an intra-chain bond in the A-chain. In the family, insulin, IGF1, and IGF2 have been reported to be involved in the regulation of glucose metabolism and signal through tyrosine kinase/growth factor receptors, which are single transmembrane receptors. Another member of the relaxin/insulin superfamily is Insulin-Like (INSL) 5 (Conklin et al. 1999, Genomics, 60(1):50-56), which is believed to be a selective ligand for GPCR142 (see, e.g., U.S. Provisional Application No. 60/580,083, the disclosure of which is incorporated by reference herein). Two other members in the family are relaxin and INSL3, ligands for LGR7 and/or LGR8, which are GPCRs with leucine-rich repeats at the N-terminal extra-cellular domain. Relaxin-3 was also reported to be an additional ligand for LGR7.
The GPCR142 expression pattern is distinct from that of GPCR135 and relaxin-3 with an abundant peripheral tissue distribution in addition to expression in the brain (Liu et al., 2003, Journal of Biological Chemistry, 278(50):50765-50770)). We have found that GPCR142 is highly conserved among human, monkey, cow, and pig, but is less conserved in the mouse, and a pseudo-gene exists in the rat despite a fair degree of conservation of relaxin-3 genes in both rodent species, suggesting that GPCR142 has a distinct function from GPCR135 and diminished function in the rodents. All the physiological functions of GPCR135 and GPCR142 remain to be fully elucidated. In vivo administration of relaxin-3 also activates LGR7, which is expressed in both the brain and periphery (Hsu et al., 2000, Mol. Endocrinol., 14:1257-1271; Hsu et al., 2002, Science, 295:671-674; Tan et al., 1999, Br. J. Pharmacol., 127:91-98). Thus, in vivo study of GPCR135 or GPCR142 functions has been confounded by the lack of selective pharmacological tools for these two receptor subtypes.